Overview

Egg Yolk Agar (EYA, McClung-Toabe modification) is the classical differential medium for the lecithinase and lipase reactions of Clostridium species. First described by McClung & Toabe (1947) Journal of Bacteriology 53:139, it remains the reference medium for clostridial phenotypic identification in food microbiology (FDA BAM ch. 16 botulinum, ch. 17 perfringens; ISO 7937:2004 C. perfringens enumeration), clinical wound microbiology (Wadsworth ch. 9), and veterinary anaerobic bacteriology.

The differential mechanism reads two distinct phospholipid-hydrolysing enzymes against a single substrate: egg yolk. Lecithinase (phospholipase C; the C. perfringens α-toxin) hydrolyses lecithin (phosphatidylcholine) to insoluble diglyceride and phosphocholine — visible as an opacity halo extending several mm beyond the colony into the egg-yolk-supplemented agar. Lipase (triglyceride esterase; characteristic of C. botulinum and C. sporogenes) hydrolyses triglycerides to free fatty acids — visible as a pearly iridescent layer on the colony surface (free fatty acids form a lipid film). C. perfringens shows both a lecithinase opacity halo AND (when blood is added) a double-zone β-haemolysis (inner θ-toxin / perfringolysin O zone + outer α-toxin lecithinase zone) — the combined picture is essentially pathognomonic.

The Nagler half-plate test (using C. perfringens type A α-toxin antitoxin spread on half a plate) is the confirmatory identification step: C. perfringens lecithinase is suppressed on the antitoxin half (no halo), while C. bifermentans, C. sordellii, and C. novyi lecithinases are not antitoxin-suppressible. For selective application, add neomycin 100 mg/L (post-autoclave filter-sterilised) to make EYA-Neo, which suppresses facultative aerobes and supports Clostridium-specific phenotyping.

We also have

YCFA Modified Medium · YCFA Full Recipe · YCFA Base · Modified Chopped Meat Broth (ATCC 1490) · Chopped Meat with Carbohydrates · Beef Granules

Package Contents

Each GMExpression Egg Yolk Agar Complete Kit contains:

  • Base agar (dehydrated) — pre-weighed McClung-Toabe base (proteose peptone 40 g/L + Na2HPO4 5 g/L + KH2PO4 1 g/L + NaCl 2 g/L + MgSO4·7H2O 0.1 g/L + glucose 2 g/L + agar 20 g/L) for 1 L final volume.
  • Vial EY — Sterile egg yolk emulsion (50 % egg yolk in saline; Oxoid SR0047 / BD BBL™ 211946 equivalent, prepared from SPF-flock eggs under Australian DAFF certification). 100 mL bottle, amber, light-protected.
  • Vial Neo (optional, EYA-Neo selective variant) — Neomycin sulphate stock 100 mg/mL filter-sterilised. Single-use 1 mL aliquot.
  • Vial Antitoxin (optional, for Nagler test) — C. perfringens type A α-toxin antitoxin, freeze-dried, single-use. CSL Biological Products / Wirrina Bioservices Australian source.
  • Instruction manual (A5 booklet, v1.0) with full McClung-Toabe 1947 protocol, FDA BAM C. perfringens / C. botulinum annex, ISO 7937 enumeration workflow, Nagler half-plate confirmation procedure, and a QC organism panel (C. perfringens ATCC 13124 positive; C. butyricum ATCC 19398 negative).

Customisation options on request: EYA Base only (without egg yolk supplement) for customers sourcing egg yolk locally — longer shelf life; EYA + 5 % defibrinated sheep blood (haemolysis + lecithinase + lipase combined differential — Wadsworth recommended primary clostridium plate); EYA-Neo pre-supplemented; TSC variant (Tryptose-Sulfite-Cycloserine, FDA BAM ch. 17 C. perfringens selective with sulphite-blackening differential); Nagler half-plate kits (10 antitoxin discs).

Composition — per 1 L equivalent unless stated otherwise

McClung-Toabe Egg Yolk Agar base (per 1 L)

ComponentConcentrationFunction
Proteose peptone40.0 gPrimary peptide source (BD Difco "Proteose Peptone No. 3"; substitutable with tryptone + soytone blend)
Disodium hydrogen phosphate (Na2HPO4)5.0 gBuffer (alkaline)
Potassium dihydrogen phosphate (KH2PO4)1.0 gBuffer (acidic)
Sodium chloride (NaCl)2.0 gOsmotic balance
Magnesium sulphate heptahydrate (MgSO4·7H2O)0.1 gTrace cofactor
D-Glucose2.0 gLimited carbohydrate — avoids excessive acidification masking the lecithinase reaction
Agar20.0 gSolidifying agent (higher than standard 15 g/L due to plate-thickness requirement)

Pre-autoclaving pH: 7.6 ± 0.2 at 25 °C (slightly alkaline; favours lecithinase activity).

Egg yolk emulsion supplement (post-autoclave addition)

ComponentConcentrationFunction
Sterile egg yolk emulsion (50 % v/v in saline) — Vial EY100 mL per litre (10 % v/v final) — typical; or 50 mL/L (5 %) for less-fatty variantCritical differential substrate — provides lecithin (lecithinase substrate) and triglycerides (lipase substrate)

Optional selective and differential variants

Variant / additional componentConcentrationEffect
Neomycin sulphate (EYA-Neo selective; Vial Neo)100 mg/L (post-autoclave)Suppresses facultative aerobes; Clostridium intrinsically resistant in relevant species
Defibrinated horse / sheep blood50 mL/L (5 % v/v post-autoclave)Haemolysis reading (C. perfringens double-zone β-haemolysis); enhanced colony morphology
D-cycloserine (TSC variant; FDA BAM ch. 17)400 mg/L (post-autoclave)More selective for C. perfringens
Ammonium ferric citrate + sodium metabisulphite (TSC variant)1.0 + 1.0 g/L (pre-autoclave)Produces black colonies from H2S + iron (alternative C. perfringens differential)
C. perfringens type A α-toxin antitoxin (Vial Antitoxin)Half-plate spread (Nagler test)Suppresses C. perfringens lecithinase on antitoxin-side only; confirmatory identification

→Culturomics and Media Navigator

Need help matching this medium to your microorganism — or starting from a target organism and finding the medium that fits? Use the GMExpression Culturomics and Media Navigator: a curated, cross-referenced tool that lets you (i) start from a medium and view the bacterial taxa it is recommended for, or (ii) start from a target organism (genus, species, or DSMZ/ATCC strain ID) and view the panel of GMExpression media that will support it. The Navigator integrates Egg Yolk Agar (McClung-Toabe modification) with the wider YCFA, Chopped Meat Broth, BHI-S, Wilkins-Chalgren, Modified GAM, Anaerobic Columbia Blood Agar, GMM, M2GSC, BBE, KVLB, CCFA, and TPY catalogue.

Use and Applications

  • Identification of Clostridium perfringens (lecithinase-positive double-opacity zone; pathognomonic when antitoxin inhibition is added in the Nagler half-plate variant). FDA BAM ch. 17 reference workflow.
  • Identification of C. bifermentans, C. sordellii, C. novyi — lecithinase-positive but not C. perfringens-antitoxin-suppressible (Nagler-negative).
  • Identification of C. botulinum, C. sporogenes, C. tetani — lipase-positive (pearly iridescent layer over colony from triglyceride hydrolysis). FDA BAM ch. 16 reference workflow for botulism investigation in foods.
  • Food microbiology — C. perfringens enumeration per ISO 7937:2004; C. botulinum detection per FDA BAM ch. 16; meat-product, dairy, and ready-to-eat-food Clostridium spoilage investigation.
  • Veterinary microbiology — Clostridium species identification in animal pathology (enterotoxaemia by C. perfringens types A–E, blackleg by C. chauvoei, malignant oedema by C. septicum, botulism by C. botulinum).
  • Wound-infection isolates — differentiation of clostridial species from clinical wound and tissue samples per Wadsworth ch. 9 algorithm.
  • Research on lecithinase / phospholipase / lipase enzymes — long-standing in-vitro enzyme assay platform.
  • Faecal microbiota transplant (FMT) donor screening — exclusion of C. perfringens and C. difficile from donor stool prior to FMT preparation.

Compatible Microorganisms

Lecithinase-positive (opacity halo surrounding colonies)

  • Clostridium perfringens (ATCC 13124) — large opacity zone; opacity suppressed by C. perfringens type A α-toxin antitoxin on a Nagler half-plate (confirmatory). Reference positive QC organism.
  • Clostridium bifermentans (ATCC 638) — lecithinase-positive; non-antitoxin-suppressible (distinguishes from C. perfringens).
  • Clostridium sordellii (ATCC 9714) — lecithinase-positive; closely related to C. bifermentans.
  • Clostridium novyi type A (ATCC 17861) — lecithinase-positive.
  • Bacillus cereus (ATCC 14579; aerobic but grows on EYA) — lecithinase-positive; common environmental / food contaminant that must be differentiated from Clostridium by aerobic-growth check.

Lipase-positive (iridescent pearly layer over colony)

  • Clostridium botulinum types A–G (ATCC 19397 type A; ATCC 25763 type B) — lipase-positive on EYA. Critical for food-microbiology botulism detection.
  • Clostridium sporogenes (ATCC 7955) — lipase-positive; commonly used as non-toxigenic surrogate for C. botulinum in food-safety challenge tests.
  • Clostridium tetani (ATCC 19406) — variable lipase reaction.
  • Some strains of C. novyi type B — lipase-positive.

Both lecithinase- and lipase-positive (rare)

  • Occasional C. sporogenes clinical isolates.

Negative for both reactions (control / differential)

  • C. tetani (type strain) — both reactions weak or negative.
  • C. ramosum / Erysipelatoclostridium ramosum — negative.
  • C. butyricum (ATCC 19398) — negative for both. Reference negative QC organism.
  • Clostridioides difficile (ATCC 9689) — negative for both. Useful as a differential check (also relevant for the CCFA-EY variant; see § C2).
  • Most non-Clostridium anaerobes — negative.

Preparation

1Use pre-poured plates (recommended). Pre-poured GMExpression EYA plates with egg yolk emulsion already incorporated and pre-reduced are released from QC ready to inoculate. Remove from foil pouch inside the AAE or just before inoculation; equilibrate at room temperature for 10 min.
2If preparing from kit — combine base. Weigh and combine base ingredients (provided pre-weighed in the dehydrated-base sachet): 40 g proteose peptone + 5 g Na2HPO4 + 1 g KH2PO4 + 2 g NaCl + 0.1 g MgSO4·7H2O + 2 g glucose + 20 g agar.
3Suspend. Combine with 900 mL distilled water (not 1000 mL — leave 100 mL headroom for the egg yolk emulsion). Soak 5 min.
4Heat-dissolve. 95–100 °C with stirring; agar takes 5–8 min to fully dissolve. Confirm clarity (no agar grains).
5Adjust pH. Target 7.6 ± 0.2 at 25 °C (slightly alkaline). The alkaline pH favours lecithinase enzyme activity in the differential reaction.
6Autoclave. 121 °C × 15 min.
7Cool to 50 °C. In a water bath. Critical: the 50 °C medium temperature is precise — adding egg yolk to medium below 45 °C causes premature gelation of agar near the egg-yolk droplets, producing uneven plates with crystal-like artifacts. Above 60 °C, egg-yolk proteins denature and lose enzyme substrate.
8Add the egg yolk emulsion. 100 mL of Vial EY (sterile 50 % egg yolk emulsion) per 900 mL of cooled base = 10 % v/v final concentration. Mix gently — avoid foaming. The emulsion is viscous; pipette slowly with a serological pipette.
9Optional supplements. EYA-Neo: 1 mL of Vial Neo (100 mg/mL neomycin sulphate, filter-sterilised) = 100 mg/L final. EYA-Blood: 50 mL of sterile defibrinated horse / sheep blood = 5 % v/v. TSC: pre-autoclave 1 g/L ammonium ferric citrate + 1 g/L sodium metabisulphite + post-autoclave 0.4 g/L D-cycloserine.
10Pour plates. 25–30 mL per 90 mm plate (the medium is thick due to egg yolk content). Pour at a level surface inside a laminar flow hood. Allow to set 30 min at room temperature. Bag and store at 4 °C, light-protected.
11For Nagler half-plate. Before inoculation, spread an aliquot (50–100 µL) of reconstituted C. perfringens antitoxin (Vial Antitoxin) on half of a poured plate; let diffuse for 30 min at room temperature. Inoculate both halves with the test strain perpendicular to the antitoxin boundary. Read for lecithinase suppression on the antitoxin half (C. perfringens shows no halo on the antitoxin half; C. bifermentans / C. sordellii show halos on both halves).

Critical control points

  • Egg yolk source. Commercial pre-sterilised egg yolk emulsion (Vial EY in the GMExpression kit) is strongly preferred over in-house preparation — consistency, sterility, and avoidance of Salmonella contamination. If in-house preparation, use eggs from a documented SPF (specific-pathogen-free) flock under Australian DAFF certification; filter-sterilise through 0.45 µm membrane (the 0.22 µm clogs); store at 4 °C and use within 14 days; or freeze at −20 °C for 12 months.
  • Temperature of egg yolk addition (50 °C critical). Below 45 °C: premature agar gelation, uneven plates. Above 60 °C: egg-yolk protein denaturation, loss of enzyme substrate. Maintain the medium at 50 °C in a water bath with magnetic stirring; add egg yolk and pour within 10 min.
  • Plate thickness. Pour to 5–6 mm depth (= 25–30 mL per 90 mm plate). Thinner plates dehydrate too quickly during the 24–48 h incubation, obscuring the differential reading.
  • Antitoxin sourcing (for Nagler test). C. perfringens type A α-toxin antitoxin is supplied as Vial Antitoxin (freeze-dried; reconstitute with sterile saline immediately before use). Australian sources: CSL Biological Products, Wirrina Bioservices, or Sigma-Aldrich. The antitoxin must be type-A-specific to give correct identification.

Cautions

Double-opacity zone of C. perfringens. The pathognomonic C. perfringens feature is a double zone of opacity: inner zone of clear haemolysis (if blood added; θ-toxin / perfringolysin O) surrounded by an outer zone of dense opacity (α-toxin / phospholipase C). On standard EYA (no blood) only the outer opacity (lecithinase) is visible. For full double-zone reading, prepare EYA + 5 % defibrinated sheep blood (add blood at the same step as the egg yolk supplement).
Egg yolk batch variability. Commercial egg yolk emulsions vary slightly in lecithin / phospholipid ratio between lots, affecting opacity-zone size. Mitigation: QC each new batch with C. perfringens ATCC 13124 (positive control) and C. butyricum ATCC 19398 (negative control). Sigma-Aldrich, BD BBL™, Oxoid SR0047, and HiMedia FD046 are interchangeable for routine work; for compliance work, validate the specific lot.
Australian biosecurity and egg sourcing. Chicken eggs are subject to Australian state-level biosafety regulation (especially Salmonella surveillance under the Egg Safety Standard). Commercial sterilised egg yolk emulsion (Vial EY in this kit) is the preferred source; in-house preparation requires SPF eggs and validated sterilisation. Do not use supermarket eggs for in-house emulsion preparation.
Lecithinase / lipase failure causes. Several possible causes for weak / absent reactions: (a) inadequate anaerobiosis — clostridial lecithinase / lipase expression is suppressed in the presence of trace oxygen; verify the anaerobic atmosphere; (b) too-fresh inoculum — reactions are best read at 48 h, not 24 h; (c) egg yolk emulsion stored too long (> 14 days post-opening at 4 °C) or freeze-thawed multiple times; (d) the isolate is genuinely a lecithinase / lipase weak-variant (some C. perfringens strains show smaller opacity zones).
Differentiation from Bacillus cereus. B. cereus (aerobic) grows on EYA and gives a lecithinase reaction that can be confused with Clostridium. Differentiation: Bacillus grows aerobically; Clostridium requires anaerobiosis. Always incubate EYA anaerobically when reading Clostridium phenotypes. For aerobic B. cereus work, use mannitol-egg-yolk-polymyxin (MYP) agar instead.
Shelf life. Prepared EYA plates: 3 weeks at 4 °C aerobic (egg yolk lipids auto-oxidise slowly, eventually compromising lipase reading); 6 weeks vacuum-sealed with oxygen absorber. Stored EYA can develop a slight rancid odour from fatty-acid oxidation — discard if odour is strong. Egg-yolk emulsion Vial EY: 14 days at 4 °C once opened; 12 months frozen at −20 °C.

Storage and Expiry · Safety

  • Pre-poured plates (sealed in foil pouches with O2 indicator): 4 °C, light-protected. Shelf life 3 weeks aerobic-sealed; 6 weeks vacuum-sealed with O2 absorber.
  • Dehydrated EYA base (powder, without egg yolk): 15–30 °C, sealed in original packaging. Shelf life 30 months (base is stable; egg yolk is the perishable component).
  • Vial EY (sterile egg yolk emulsion): 4 °C, light-protected. Shelf life 12 months sealed; use within 14 days of opening (egg-yolk lipids slowly auto-oxidise). For longer storage: freeze at −20 °C in aliquots; stable 12 months frozen.
  • Vial Neo (Neomycin): 4 °C for 1 month; −20 °C for 6 months.
  • Vial Antitoxin (C. perfringens type A antitoxin, freeze-dried): 4 °C, sealed under N2. Shelf life 24 months freeze-dried; reconstitute with sterile saline immediately before use; reconstituted antitoxin: 24 h at 4 °C only.
  • Prepared plates, opened pouch: use within 24 h once oxygen-exposed.

Safety notes. Egg yolk is an allergen — handle with care in laboratories with personnel with egg allergies; clean spills promptly. Antitoxin is a biological product (equine origin for most commercial preparations) — handle as Class 2 biological; some individuals develop hypersensitivity to equine serum proteins (Type I or Type III). C. perfringens, C. botulinum, and other Clostridium spp. handled on this medium are Risk Group 2 organisms (some C. botulinum activities Risk Group 3 — botulinum toxin is a Select Agent) — comply with applicable Australian OGTR and state-level biosafety regulations. SDS available on request.

References

  1. McClung LS, Toabe R. (1947). The egg yolk plate reaction for the presumptive diagnosis of Clostridium sporogenes and certain species of the gangrene and botulinum groups. Journal of Bacteriology 53(2): 139–147. [Primary reference]
  2. Stickland LH. (1934). The chemical reactions by which Cl. sporogenes obtains its energy. Biochemical Journal 28: 1746–1759. (Stickland reaction; relevant to clostridial metabolism on EYA.)
  3. Jousimies-Somer HR et al. (2002). Wadsworth-KTL Anaerobic Bacteriology Manual, 6th ed., chapter 9.
  4. Cumitech 3B. (2010). Methods for the recovery of obligately anaerobic bacteria from clinical specimens. ASM Press. (EYA in Clostridium identification.)
  5. FDA Bacteriological Analytical Manual (BAM). Chapter 16 (Clostridium botulinum) and chapter 17 (Clostridium perfringens).
  6. ISO 7937:2004. Microbiology of food and animal feeding stuffs — Horizontal method for the enumeration of Clostridium perfringens — Colony-count technique.
  7. Hauschild AHW, Hilsheimer R. (1974). Evaluation and modifications of media for the enumeration of Clostridium perfringens. Applied Microbiology 27(1): 78–82. (TSC modifications.)
  8. BD BBL™ Egg Yolk Agar / Anaerobic Blood Agar with Egg Yolk product information 221728; Oxoid Manual 9th ed., PB0530 (Egg Yolk Emulsion).

Frequently Asked Questions

Q1. Should I order the EYA Complete Kit (with egg yolk) or the Base only?
The Complete Kit (with Vial EY sterile egg yolk emulsion) is recommended for most users — egg yolk is the perishable, supply-chain-sensitive component and the kit ensures known supplement quality. The Base-only SKU is appropriate for laboratories that already source filter-sterilised egg yolk emulsion locally (e.g., Oxoid SR0047, BD BBL™ 211946) or operate at high throughput where multiple Base sachets are combined with bulk egg-yolk emulsion.
Q2. Can EYA be used as a primary isolation medium without other plates?
No — EYA is a differential medium, not a primary isolation medium. Use it alongside non-selective primary plates (anaerobic CBA) and selective plates (BBE, KVLB, CCFA, depending on suspected pathogen) per the Wadsworth Manual workflow. EYA is read after primary growth is established (typically Day 2 of the anaerobic culture workflow), as a phenotyping step for Clostridium identification.
Q3. What is the difference between EYA and CCFA-EY?
EYA is the standalone egg-yolk + glucose + peptone differential medium (no antibiotic selectivity). CCFA-EY is CCFA (cycloserine + cefoxitin selective for C. difficile) supplemented with egg yolk for additional differential reading — used specifically in C. difficile isolation and exclusion of false-positive lecithinase-positive Clostridium colonies (which are likely C. sordellii or C. bifermentans, not C. difficile). C. difficile is lecithinase- and lipase-negative on both EYA and CCFA-EY.
Q4. How long should I incubate EYA plates?
Anaerobic, 35–37 °C, 24–48 h for primary phenotype reading (lecithinase opacity and lipase iridescence both visible by 48 h). For doubtful or slow-growing isolates, extend to 72–96 h. Beyond 96 h the egg yolk component dehydrates and the differential reading is compromised — discard plates after 96 h.
Q5. What is the purpose of including blood in some EYA variants?
Blood supplementation enables haemolysis reading alongside the lecithinase / lipase differential. C. perfringens shows the characteristic double-zone β-haemolysis (inner θ-toxin lytic zone) surrounded by the outer lecithinase opacity zone — the combined picture is essentially pathognomonic for C. perfringens. Without blood, only the outer lecithinase opacity is visible. The Wadsworth Manual recommends EYA + 5 % defibrinated sheep blood + 100 mg/L neomycin as the primary plate for suspect-Clostridium clinical specimens.
Q6. Why is the lecithinase or lipase reaction sometimes weak or absent?
Several possible causes: (a) inadequate anaerobiosis — Clostridium lecithinase / lipase expression is suppressed in trace oxygen; verify the anaerobic atmosphere; (b) too-fresh inoculum — read at 48 h, not 24 h; (c) egg yolk emulsion stored too long (> 14 days post-opening at 4 °C) or freeze-thawed multiple times; (d) the isolate is a lecithinase / lipase weak-variant. Confirmatory: spot the suspect colony on a freshly-prepared QC plate alongside C. perfringens ATCC 13124.
Q7. Can EYA be prepared without antibiotics?
Yes — the McClung-Toabe original formulation has no antibiotic; antibiotics (neomycin for selective variant; cycloserine + cefoxitin for the CCFA-EY C. difficile variant) are optional additions. For pure differential reading on a pre-identified pure culture, the non-selective EYA is sufficient. The selective variants are needed only when working with mixed primary specimens.
Q8. Is EYA compatible with the GMExpression Anaerobic Preparation Kit (APK)?
Yes — EYA plates can be pre-reduced and vacuum-sealed using the APK workflow identically to YCFA. For optimal lecithinase / lipase reading, pre-reduction for 24 h before inoculation is recommended (suppresses the oxidising bias that would otherwise inhibit expression of these clostridial enzymes). The GMExpression EYA Complete Kit is supplied pre-reduced and APK-compatible.